EA037848B1 - Fusion protein, polynucleotide, genetic construct, producer, preparation for regeneration of cartilage (variants) - Google Patents

Abstract

The invention relates to molecular biology, biotechnology, medicine, veterinary science. The invention is proposed: a fusion protein comprising a ligand to MATN1 protein, and a growth factor of EGF, TGF, FGF, IGF, the construct components are connected via a flexible link, characterized by the amino acid sequence of Fig. 1; such fusion protein further comprising the Fc-fragment of an antibody or a polypeptide binding with FcRn and/or transferrin or a fragment thereof, the construct components connected via a flexible link, characterized by the amino acid sequence of Fig. 1; encoding polynucleotide, codon-optimized for expression in the producer cells or a target organism, a genetic construct for the synthesis of the fusion protein in producer cells, or cells of a target organism, the fusion protein producer, a producer of a genetic construct based on a bacterial cell, a preparation for the regeneration of cartilage containing at least one fusion protein or genetic construct as the active agent, in an effective amount, and a physiologically acceptable carrier or buffer solution, for parenteral or, in the case of the preparation based on at least one fusion protein containing the transport domain, oral administration, in the latter case the preparation is enclosed in an enteric coating, all variants.

Description

The group of inventions relates to molecular biology, biotechnology, medicine, veterinary medicine and can be used for cartilage regeneration.

Cartilage is a type of connective tissue; Cartilage is either a part of the bone that helps to ensure its mobility, or a separate anatomical structure outside the skeleton. Articular cartilage, intervertebral discs, ear, nose, and pubic symphysis cartilages are in direct connection with the bone. Separate anatomical formations constitute a group of cartilage of the airways (larynx, trachea, bronchi), the stroma of the heart. Cartilage performs integrative-buffer, amortization, shape-supporting functions, is involved in the development and growth of bones. Biomechanical functions are carried out due to the elastic properties of the cartilage.

Diseases and problems associated with cartilage tissue can be roughly divided into three large groups. The first group includes diseases that may be associated with developmental defects (for example, ankylosis, dysplasia) (NS Kosinskaya, Disorders of the development of the osteoarticular apparatus, ed.Meditsina, 1966). The second group includes diseases caused by degenerative changes in cartilage with aging and many dystrophic processes (for example, osteoarthritis, osteochondrosis), metabolic disorders (for example, osteoporosis, gout, arthrosis, Kashin-Beck disease, ochronosis), as well as systemic diseases (rheumatism , rheumatoid arthritis, systemic lupus erythematosus, scleroderma, etc.). The third group includes damage to cartilage tissue that occurs as a result of the action of physical (mechanical, thermal, etc.), chemical and other traumatic agents (for example, perichondritis, transchordal fracture, cartilage damage due to microtraumas) (Multivolume Guide to Surgery, ed. V.V. Petrovsky, t. 5, M. -L., 1960.).

WHO statistics show that 80% of the working-age population from 30 to 50 years old suffers from various diseases of the musculoskeletal system. According to official statistics, tens of millions of people suffer from various diseases of the cartilage tissue, and even more of those who do not seek help from specialists. For example, the number of reported cases of arthritis has almost doubled over the past 20 years, and it looks like this trend will increase every year. If we interpret the statistics, it turns out that every year 1% of Russians are diagnosed with arthritis and the number of healthy Americans is declining by about the same amount. Only arthrosis, arthritis and osteoporosis account for approximately 3% of the population in Russia or the United States. About 4% of the world's population suffers from osteoarthritis, and in 10% of cases it is osteoarthritis that causes disability. According to various experts, up to 85% of the adult population of Russia is faced with this disease. In addition, according to WHO data in 2009, about 20-50 million injuries were registered in the world, which led to various problems with the musculoskeletal system or caused disability, and data for 2013 indicate that up to 500 people suffer from spinal injuries every year. thousand people (http://spinet.ru/public/dinamika_rasprostraneniy_oda.php). It should also be noted that the treatment of cartilage is the main problem in sports traumatology: damage to the cartilaginous tissue causes retirement from sports, since its restoration is currently extremely difficult to achieve with minor injuries, and with serious injuries it is impossible.

Currently, with damage to cartilage, pain relief agents are most often used - for the treatment of symptomatic pain, - non-steroidal anti-inflammatory drugs (NSAIDs), which are used orally or topically in the form of gels (Zupanets I.A. et al. Pharmaceutical care: symptomatic treatment joint and muscle pain, Pharmacist, 2002, issue 12). However, it is important to restore the structure of the cartilage, pain syndrome is a secondary phenomenon in case of damage. To restore the integrity of the cartilage, chondroprotective drugs based on chondroitin sulfate and glucosamine are currently used. However, a long course of several months of daily intake is required to improve the condition, and these drugs are effective only with minor damage to the cartilage. These drugs only stimulate the synthesis of matrix components by existing cells. However, according to the authors, the number of cells plays a key role in the restoration of cartilage. Since normally, and even more so when cartilage is damaged, these cells are present in small quantities, then influencing them to intensify the synthesis of matrix components will only slightly improve the condition of the cartilage.

Known invention described in US 9133259 B2 (19.04.2012), which relates to protein complexes containing oligomeric cartilage matrix protein (COMP), combined with one or more growth factors (for example, or TGFe1, or FGF, or both of these factors) to stimulate chondrogenesis and / or osteogenesis and restore bone and cartilage structures. In one embodiment, the growth factor is TGFe1 or different classes of BMP (bone morphogenetic protein). In the description of the invention, it is disclosed that in the most preferred embodiment, the protein complex is located within a self-absorbable matrix, which is a plate or bone graft. The matrix additionally contains collagen or fibrin. To achieve a therapeutic effect, protein complexes must be brought into contact with the affected area of cartilage or bone or implanted in them. However, this is an invasive and time-consuming method with complications.

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The authors of the present invention proposed a hybrid protein (variants) comprising a ligand to the MATN1 protein and an EGF protein, either TGF, or FGF, or IGF, the components of the construct are connected by a flexible bridge.

MATN1 (matrilin) is a cartilage matrix protein. Mutations in the gene encoding this protein are associated with various hereditary chondrodysplasias and idiopathic scoliosis (http://www.ncbi.nlm.nih.gov/gene?Db=gene&Cmd=ShowDetailView&TermToSeamch=4146, Montanaro L. et al., Evidence of a linkage between matrilin-1 gene (MATN1) and idiopathic scoliosis. Scoliosis 1: 21, 2006)

EGF (Epidermal Growth Factor) is an epidermal growth factor, a protein that stimulates cell growth and cell differentiation of the epithelial cover. This protein acts by binding to the epidermal growth factor receptor on the cell surface, which stimulates the activity of intracellular tyrosine kinases (Dawson JP et al Epidermal growth factor receptor dimerization and activation require ligand-induced conformational changes in the dimer interface, Mol. Cell. Biol. 25: 17, 2005).

TGF (Transforming growth factor) is a transforming growth factor consisting of two classes of polypeptide growth factors, TGFa and TGF-β. TGFa induces the development of epithelial tissue. TGF-β is represented by three subtypes TGFe1, TGFe2 and TGFe3. These proteins play a decisive role in tissue regeneration, cell differentiation, and embryonic development. TGFs act by binding to receptors, which in turn phosphorylate specific signaling effectors, causing signaling protein complexes to form. The resulting complexes are translocated from the cytoplasm to the nucleus, where they regulate the transcription of specific genes (Matt. P et al., Circulating transforming growth factor- {beta} in Marfan syndrome. Circulation 120 (6): 526-32, 2005).

FGFs (Fibroblast growth factor) - fibroblast growth factors. It is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. It has been shown that the signaling of fibroblast growth factors requires interactions with proteoglycans located on the cell surface. Fibroblast growth factors play a key role in the processes of proliferation and differentiation of a wide range of cells and tissues (https://embmyology.med.unsw.edu.au/embrnyology/index.php/Developmental_Signals_-_Fibroblast_Growth_Factor; http://dic.academic.ru /dic.nsf/rawiki/188282).

IGF - Insulin-like human growth factor of the first type (IGF-1, IGF-1), a peptide growth factor that mediates the growth-stimulating effects of somatotropic hormone (STH), is a polypeptide consisting of 153 amino acids, with a molecular weight of about 17 kDa. Under the influence of STH, IGF-1 is formed in the liver and other tissues. IGF-1 stimulates the proliferation of cells in most tissues, primarily bone, cartilage and muscle. IGF-1 is an anabolic agent that has been shown to improve metabolism (Meyer N.A. et al. 1996. J. Trauma 31 (6): 1008-1012) after various types of injury. IRF-1 treatment improves tissue healing (Martin, P. 1997. Science 276: 75-81. 1997; Steenfos H. 1994. Scand J Plast Reconstr Hand Surg 28: 95-105; Pierre et al. 1997. J Bum Care Rehab 18 (4): 287-291).

The hybrid protein proposed by the present inventors is tropic to the cartilage matrix, due to the fact that it contains a ligand to the cartilage matrix protein MATN1 (matrilin), respectively, it can be administered both parenterally and systemically, which facilitates its use. This protein also contains a growth factor from EGF, or TGF, or FGF, or IGF. The components of the fusion protein are connected by a flexible bridge, which makes it possible to achieve the functioning of each protein component.

The technical result from the use of a hybrid protein or a preparation based on it according to the invention (variants) is expressed in the simplification of the treatment of cartilage damage. This is achieved due to the absence of the mandatory need for the introduction of the protein or drug locally, due to targeted delivery due to the use in the structure of the hybrid protein of an element that binds exclusively to the protein of cartilage tissue, and it is proposed by the authors. Also, this technical result is achieved with local administration due to a simpler form of administration, injection, without the need to use a matrix of a special composition, which must be previously designed.

The technical result from the use of a fusion protein, or a preparation based on it according to the invention (variants) is expressed in an increase in safety and, accordingly, in a decrease in complications from the therapy of cartilage damage due to the use of safer instruments for delivering the fusion protein to the cartilage.

The technical result from the use of a hybrid protein, or a preparation based on it according to the invention (variants) is expressed in an increase in the effectiveness of treatment of cartilage damage, due to a more uniform distribution of the hybrid protein in the cartilage tissue, due to the tropism to the matrix protein, in contrast, according to the prototype, clearly defined the injection site when administered as part of a matrix or even injected.

The technical result from the use of a hybrid protein or a preparation based on it according to the invention (variants) also consists in expanding the spectrum of active substances or preparations, respectively, to restore the structure of the cartilage. In case of contraindications to the use of analogs, or unwillingness to use analogs due to their above-described disadvantages, this protein, or a pre-2 037848 paratha, which has a different principle of operation, will allow for the regeneration of cartilage. Due to the fact that the problem of cartilage regeneration is very acute, and not many people succeed in bringing the drug to the market, the proposed invention will increase the chances in the fight against this widespread ailment.

The authors of the present invention also proposed a genetic construct (variants) for the expression of a hybrid gene in the cells of a producer or a target organism (variants), encoding a hybrid protein (variants) comprising a ligand to the MATN1 protein and an EGF protein, or TGF, or FGF, or IGF, connected by a flexible bridge. Also proposed is a preparation based on 1 of such a construct for parenteral administration. Such a construction and a preparation based on it are not known in the art. The advantages and technical result of the specified genetic construct and the preparation based on it are the same as for the fusion protein, but there are also additional advantages.

So the technical result also consists in increasing the duration of the effect and is achieved by using a genetic construct, with which, after introduction into the body, a hybrid protein is synthesized; and also by the fact that this genetic construct contains elements that determine the stability of mRNA and, accordingly, increase the half-life of mRNA, as a result, protein synthesis from one mRNA molecule is carried out more times, and as a result, the amount of synthesized protein increases; and also by the fact that the nucleotide sequence of the hybrid gene is codon optimized for expression in the target organism, as a result, protein synthesis is more intensive. When introduced into practice, this will significantly reduce the amount of injected plasmid DNA (10-50 times), compared with the doses currently used in domestic and world practice for gene therapy.

In addition, the technical result is to increase safety. The specified technical result is achieved by the fact that the protein synthesized in the body from a genetic structure undergoes natural post-translational processing, and also ensures the correct folding of the protein due to cellular chaperones. The specified technical result is also achieved due to the presence in the genetic construct of such regulatory sequences as a silencer and / or insulator, in one of the embodiments of the invention, due to which the amount of synthesized protein is also controlled, and, in principle, protein synthesis as such, and , respectively, over the action of the drug: if necessary, it is possible to quickly stop or reduce the expression of the gene. In the latter variant, tissue-specific expression is also possible, if necessary.

The technical result also consists in simplifying and cheapening the production of a regenerative preparation by avoiding the production and purification processes of protein preparations in vitro, due to the fact that protein synthesis occurs in vivo. The production, purification and storage of DNA preparations are more economically profitable than protein preparations, since the former are more stable, they can be produced in large quantities at lower costs.

Obtaining a drug for the restoration of cartilage structures, which would be convenient for administration, is an important task of modern medicine and veterinary medicine. The oral route of drug administration is the simplest, safest and most common. Mainly, this method is used to prescribe medication for drugs that are well absorbed by the mucous membrane of the stomach or intestines. The therapeutic level of the drug in the blood is reached within 30-90 minutes after its administration and lasts for 4-6 hours, depending on the properties of the active ingredient and the composition of the drug. The advantages of this method of administration are the possibility of administering various dosage forms (powders, tablets, pills, dragees, decoctions, mixtures, etc.), the simplicity and availability of the method; also, the method does not require compliance with sterility. The disadvantages of oral administration of drugs include a slower, than with other methods of taking drugs, the development of a therapeutic effect; the speed and bioavailability are individual for each patient, since they are influenced by food, the organic and functional state of the gastrointestinal tract, and other medications; oral administration is ineffective for drugs that are poorly absorbed or destroyed in the digestive tract; also oral administration is difficult or impossible with vomiting and when the patient is unconscious (http://sestrinskoe-delo.ru/puti-isposobi-primeneniya-lekarstvennich-sredstv).

From the document WO 2013059885A2 (02.05.2013) polypeptide constructs are known containing a peptide or polypeptide signal ligand associated with an antibody or its antigen-binding part. The serum half-life of antibodies and biodistribution can be altered based on modification of the interaction between the antibody and the neonatal Fc receptor (FcRn). The document does not provide information on the possibility of oral administration of such constructs.

In documents RU 2562232 C2 (03.11.2009) and RU 2489423 (02.02.2007) analogs of the DNA alkylating agent CC-1065 and their conjugates for selective delivery and / or controlled release of one or more DNA alkylating agents are proposed. These types of conjugates are proposed by the inventors for the treatment of diseases caused by unwanted cell proliferation (tumor diseases). Conjugates can include various proteins, for example trans-3 037848 ferrin, or epidermal growth factors (EGF), or interleukins (IL-2, IL-6), or transforming growth factors (TGF), such as TGF-α and TGF-β , or other. Also, part of the conjugate is an antibody having modifications in amino acid residues that interact with Fc receptors. The conjugate elements are linked together by a linker, for example, one that includes a triazole group; a single bond (-C (O) -, -O-, -S-) can also be a linker. Preferably, the linker is small (a linker having no more than 4 bonding atoms). The pharmaceutical compositions containing the conjugates are proposed by the inventors to be administered by various routes (oral administration, topical administration or by injection). However, non-specific delivery of the conjugate and its penetration into non-target cells is possible.

From the document US 8932589 (B2) (03/12/2013) compositions are known containing a fusion protein, which includes the Klotho protein or a fragment thereof, FGF or a fragment thereof, and possibly a modified Fc fragment that increases the affinity or half-life in serum. Protein Clotho or its active fragment is able to bind to the FGF receptor. The components of the fusion protein can be linked to each other by a peptide bond or via a linker. The compositions are used to treat metabolic disorders and age-related diseases (including diseases such as osteoporosis and osteoarthritis). The compositions are presented in solid, liquid or aerosol form, and the compositions can be administered in a variety of ways, incl. orally, but the most preferred is intravenous administration - drip or bolus injection. It should be noted that the Clotho protein is not specific for cartilage tissue.

The fusion protein proposed by the authors of the present invention is tropic to the cartilage matrix, due to the fact that it contains a ligand to the cartilage matrix protein MATN1. This protein also contains a growth factor from EGF, or TGF, or FGF, or IGF, as well as an Fc fragment of an antibody, or a polypeptide that binds to FcRn for parenteral or oral delivery. The components of the fusion protein are connected by flexible bridges, which makes it possible to achieve the functioning of each protein component.

The technical result from the use of a fusion protein, or a preparation based on it according to the invention (variants), is expressed in an increase in the effectiveness of the treatment of cartilage damage, since the fusion protein works only in cartilage tissue. This is achieved due to the effect of the fusion protein, or a preparation based on it exclusively on the cartilaginous tissue, through the use of the ligand for matrilin proposed by the authors.

The technical result from the use of a fusion protein, or a preparation based on it according to the invention (variants), is expressed in an increase in safety due to a decrease in complications, the absence of unpredictable results from the therapy of cartilage damage due to the effect of a fusion protein, or a preparation exclusively on cartilage tissue, due to the use of proposed by the authors of the ligand to matrilin.

The technical result from the use of a hybrid protein, or a preparation based on it according to the invention (variants), also consists in expanding the spectrum of active substances or preparations, respectively, to restore the structure of the cartilage.

In documents US 2011092677 (A1) (04.21.2011) and WO 2004020404 (A2) (03.11.2004) a transtele is described containing one or more variable domains, or CDRs of antibodies to one or more different antigens for the treatment or prevention of diseases, as well as transferrin, or modified transferrin, which can treat some forms of arthritis when used as antigen (s) TNFa. The antigen (s) can (gut) be TGF or EGF or FGF, then the use will be different, but it is not specified in the document. The other antigens mentioned in the document are targets for transtel blocking. Preferably, said modified transferrin groups do not undergo glycosylation or practically do not undergo glycosylation, do not bind iron and / or the transferrin receptor, which positively affects the half-life. Also provided are compositions containing the above transtels. The compositions are administered in a variety of ways, parenterally or orally. These transtels for the treatment of arthritis act by binding and thus blocking TNFa, and the transtels are not targeted to any tissue. Thus, they do not induce cartilage regeneration. Transtels that bind to said growth factor (s) are intended to block the target. In the case of the proposed by the authors of the present invention, the principle of using the growth factor is different.

The fusion protein proposed by the authors of the present invention is tropic to the cartilage matrix, due to the fact that it contains a ligand to the cartilage matrix protein MATN1. The fusion protein also contains a growth factor from EGF, or TGF, or FGF, or IGF, as well as transferrin, or a fragment thereof, for parenteral administration or for oral delivery. The components of the fusion protein are connected by flexible bridges, which makes it possible to achieve the functioning of each protein component.

The technical result from the use of a fusion protein or a preparation based on it according to the invention (variants) is expressed in the regeneration of cartilage by targeting the fusion protein to the cartilage matrix, as well as attracting there and stimulating the growth of the required cells, due to the use of a growth factor in the fusion protein structure.

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The technical result from the use of a fusion protein, or a preparation based on it according to the invention (variants) is expressed in an increase in safety due to a decrease in complications, the absence of unpredictable results from the therapy of cartilage damage due to the action of a fusion protein, or a preparation exclusively in cartilage tissue, due to the use of the proposed the authors of the ligand to matrilin.

The technical result from the use of a fusion protein or a preparation based on it according to the invention (variants) also consists in expanding the range of active substances or preparations, respectively, for the treatment of cartilage damage, which can be administered orally.

The invention is described in US 9133259 B2 (19.04.2012), which relates to protein complexes containing oligomeric cartilage matrix protein (COMP) combined with one or more growth factors, for example, either TGFe1, or FGF, or both of these factors, to stimulate chondrogenesis and / or osteogenesis and restoration of bone and cartilage structures. However, unlike the claimed invention, this application does not offer oral administration of drugs containing the aforementioned protein complexes. Thus, the description of the invention discloses that in the most preferred embodiment, the protein complex is located within a self-absorbable matrix, which is a plate or bone graft. The matrix additionally contains collagen or fibrin. To achieve a therapeutic effect, protein complexes must be brought into contact with the affected area of cartilage or bone or implanted in them, which is an invasive and time-consuming method with complications.

The document WO 2004/062602 A2 (29.07.2004) proposes compositions and methods for the targeted delivery of substances to polarized cells (intestinal epithelial cells) for therapeutic, prophylactic or diagnostic purposes. The compositions contain a construction of several elements: an element for binding to the surface of a polarized cell; an element that allows the composition to penetrate into the cell; an element, polypeptide, or chemical moiety that is actually used for therapeutic, prophylactic, or diagnostic purposes. For example, if the composition is used for diagnostic purposes, the third element can be a contrast agent, for therapeutic purposes, an antibody or an antibody fragment. The first two elements can be a polypeptide, antibody or antibody fragment that can bind, for example, a transferrin receptor or FcRn. Structural elements are covalently or non-covalently bonded to each other. The compositions are intended for oral administration. However, nothing has been said about additional elements that allow targeted delivery of molecules that have passed through the epithelial layer. In the text, only the delivery of molecules through the epithelial layer is described; the further route of the introduced molecules is not disclosed. Accordingly, the third structural element used mediates the fact that the structure either remains in the epithelial cell, or is carried throughout the body and has a systemic effect.

The idea of creating constructs or fusion proteins, including or binding to transferrin or modified transferrin, as well as comprising an antibody Fc fragment or a moiety that binds to FcRn, i.e. such constructs or fusion proteins contain both transferrin and an Fc fragment, for various purposes, for example, improving the properties of already existing proteins, for creating transport proteins, for obtaining highly effective therapeutic agents, etc., is discussed in documents US 8173860 B2 (05/08/2012) , WO 2001/046254 A1 (06/28/2011), WO 2012/116453 A1 (09/07/2012), WO 2015/070014 A1 (05/14/2015), WO 2012/170969 A2 (12/13/2012). Oral administration is contemplated for all of them, except as described in the first document. Such constructs are preferably administered parenterally. There are no drugs for cartilage regeneration among them.

In documents RU 2555532 C2 (06.11.2009) and RU 2490278 C2 (19.12.2008) various variants of antibodies specific for IL-1R1, which is expressed on various types of cells, or α-receptor of interleukin-4 (hIL-4Ra) are presented person. The claimed antibodies may be useful for the treatment of IL-1R1 mediated disorders, including also rheumatoid arthritis, but the key diseases that the authors propose to treat with these antibodies are asthma and COPD (chronic obstructive pulmonary disease). These antibodies contain a framework and one or more variable loops in which the amino acid sequence of the loop or loops is mutated to create an antigen-binding site that binds to the target antigen. Such constructs include transferrin, and the binding site contains a variant Fc region having increased binding to the Fc receptor FcRn to increase serum half-life. Compositions containing these constructs are administered in various ways, incl. orally in the form of a tablet or powder, but the parenteral route of administration is preferred. This invention makes it possible to slow down the course of the disease, since the proposed antibodies block a molecule that mediates the development of rheumatoid arthritis. However, it is unable to induce cartilage regeneration in the required volume due to the fact that it does not affect the number of cells synthesizing matrix substances.

From the document US 8932589 (B2) (03/12/2013) compositions are known containing a fusion protein, which includes Kyoto protein or a fragment thereof, FGF or a fragment thereof, and possibly a modified Fc fragment that increases the affinity or half-life in serum. The Kyoto protein or its active fragment is capable of binding to the FGF receptor. The components of the fusion protein can be linked to each other by a peptide bond or via a linker. The compositions are used to treat metabolic disorders and age-related diseases, including diseases such as osteoporosis and osteoarthritis. The compositions are presented in solid, liquid or aerosol form, and the compositions can be administered in a variety of ways, incl. orally, but the most preferred is intravenous administration - drip or bolus injection. It should be noted that the Kyoto protein is not specific for cartilage tissue, therefore, therapy using this fusion protein is not targeted.

The fusion protein proposed by the authors of the present invention is tropic to the cartilage matrix, due to the fact that it contains a ligand to the cartilage matrix protein MATN1. This protein also contains a growth factor from EGF, or TGF, or FGF, or IGF, as well as transferrin or a fragment thereof, as well as an Fc fragment of an antibody, or a polypeptide that binds to FcRn, for parenteral administration or for oral delivery.

The components of the fusion protein are connected by flexible bridges, which makes it possible to achieve the functioning of each protein component.

The technical result from the use of a fusion protein or a preparation based on it according to the invention (variants) is expressed in an increase in the efficiency of cartilage regeneration by targeting the fusion protein to the cartilage matrix. Thus, the agent accumulates in the target tissue and is present there in an increased concentration, in contrast to the drug simply circulating in the bloodstream. Also, in one of the options, other growth factors are used - EGF, or TGF, or IGF.

The technical result from the use of a hybrid protein, or a preparation based on it according to the invention (variants) is expressed in an increase in safety, due to the targeting of its effect, which excludes unforeseen reactions and side effects in the body.

The technical result from the use of a fusion protein or a preparation based on it according to the invention (variants) also consists in expanding the range of active substances or preparations, respectively, for the treatment of cartilage diseases, which can be administered parenterally or orally.

The authors of the present invention also proposed parenteral administration of the above-described hybrid protein (s) containing (their) transport domain (transferrin / transferrin fragment and / or Fc antibody fragment / polypeptide that binds to FcRn), together with a hybrid (i) protein (s) not containing the transport domain, also in the composition of the preparation for cartilage regeneration, in one embodiment. In this case, the technical result coincides with that of the use of such active substances. These proteins also last longer in the body.

The technical result from the use of a preparation based on 2 hybrid proteins that do not contain a transport domain coincides with that from the use of such active substances.

The authors of the present invention also proposed a genetic construct (variants) for expression in cells of a target organism (variants), encoding a fusion protein (variants), including a ligand to the MATN1 protein, growth factor from EGF, or TGF, or FGF, or IGF, Fc-fragment antibodies, or a polypeptide that binds to FcRn and / or transferrin, or a fragment thereof; the components are connected by flexible bridges. The authors propose parenteral administration of this genetic construct, from 1 pc. in the preparation, in one of the variants - together with the genetic construct described above, which does not contain the transport domain, from 1 pc. in the preparation. Such a construction, as well as a preparation based on it, including with the addition of the above additional component (s), are not known from the prior art. The advantages and technical result are the same as for the hybrid proteins it encodes, described above, as well as the genetic construct described above, which does not contain the transport domain, and a drug based on it.

Thus, there are currently no known drugs based on hybrid protein (s) targeted to cartilage tissue for the treatment of cartilage diseases by inducing cartilage tissue regeneration, both parenterally and orally administered. Also, no drugs are known based on the genetic construct (s), with which such hybrid (s) protein (s) are synthesized in the cells of the target organism, which are administered parenterally.

Accordingly, the inventions used to obtain the above mentioned hybrid proteins are not known either: polynucleotides, genetic constructs, producers; genetic constructs: polynucleotides, producers. The technical result from them is to obtain proteins, genetic structures, respectively, and preparations according to the invention.

The essence of the invention

The urgent task of the present time is the development of drugs that would allow to achieve significant regeneration of damaged cartilaginous tissue, and which would act in a targeted manner and would be convenient to use, as well as safe and economical in production and use. This problem is solved by the proposed group of inventions (variants).

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The proposed group of inventions (variants): (1) a fusion protein comprising a ligand to the MATN1 protein, characterized by the amino acid sequence SEQ ID NO: 1, and a growth factor from EGF, TGF, FGF, IGF, characterized by the amino acid sequence SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, respectively, the components of the construct are connected by a flexible bridge characterized by the amino acid sequence SEQ ID NO: 6; (2) fusion protein (1), additionally containing an antibody Fc fragment, characterized by the amino acid sequence SEQ ID NO: 7, or a polypeptide that binds to FcRn, characterized by the amino acid sequence SEQ ID NO: 8, and / or transferrin, characterized by the amino acid sequence SEQ ID NO: 9, or a fragment thereof, characterized by the amino acid sequence SEQ ID NO: 10, the components of the construct are connected by a flexible bridge, characterized by the amino acid sequence SEQ ID NO: 6; (3) a polynucleotide encoding a fusion protein (1) or (2), codon-optimized for expression in the cells of the producer or a target organism, (4) a genetic construct for the synthesis in the cells of the producer or a target organism of a fusion protein (1) or (2 ), including a polynucleotide (3) and elements allowing to realize the indicated purpose, (5) a producer of a hybrid protein (1) or (2), (6) a producer of a genetic construct (4) based on a bacterial cell, a preparation for cartilage regeneration containing at least one / one (7) fusion protein (1) or (2), or (8) genetic construct for its / their synthesis in the cells of the target organism as an active agent, in an effective amount, as well as a physiologically acceptable carrier or buffer solution , for parenteral or (9) in the case of a preparation based on one fusion protein (2), oral administration, in the latter case, the preparation is enclosed in an enteric coating.

Detailed description of the invention

A group of inventions is proposed: a hybrid protein, a polynucleotide, a genetic construct, a producer, a preparation for cartilage regeneration (variants).

Fusion proteins are proposed, each comprising a ligand to the MATN1 protein and a growth factor from EGF, TGF, FGF, IGF, the components of the construct are connected by a flexible bridge, the sequences of the components are shown in FIG. 1. Fusion proteins are also proposed, which additionally contain from one component of an antibody Fc fragment, a polypeptide that binds to FcRn, transferrin, or a fragment thereof, the components of the construct are connected by a flexible bridge, the component sequences are shown in FIG. one.

The proposed polynucleotides encoding the described hybrid proteins, codon optimized for expression in the cells of the producer or target organism. Target organism - an organism that requires cartilage repair. With the knowledge of the amino acid sequence of the protein, one skilled in the art will be able to obtain the nucleotide sequence. Codon optimization is carried out independently, using information on the frequency of occurrence of codons from the producer, for example, in a database [for example, Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res. 2000 Jan 1; 28 (1): 292], or using specialized programs, for example, presented on the site http://www.encorbio.com/protocols/Codon.htm, or http://molbiol.ru/scrnipts/ 01_19.html.

Genetic constructs are proposed for expression in the cells of the producer or the target organism of the described polynucleotides; each construct includes, in addition to the polynucleotide, elements allowing the indicated purpose to be realized.

Under a genetic construct for expression in cells of a target organism is meant a short linear construct, or a recombinant vector, in circular or linear form, which can be a plasmid or viral vector.

A genetic construct that provides for the synthesis of a fusion protein in producer cells should contain, in addition to the fusion gene encoding a protein according to the invention, elements for maintaining and amplifying, preferably in large quantities, and efficiently functioning as intended, as well as for selecting a transformant.

A genetic construct that provides for the synthesis of a fusion protein in the cells of a target organism, a recombinant vector, should contain, in addition to the fusion gene encoding the protein of the invention, elements for maintaining and amplifying and efficiently functioning as intended.

It is most economically profitable to produce such genetic constructs in prokaryotic cells, mainly bacterial cells. In this regard, such genetic constructs according to the present invention contain elements for the maintenance and amplification, preferably in large quantities, in bacterial cells. Such essential elements are the prokaryotic origin of replication, to maintain a medium, preferably high, copy number in a cell, and a gene for the selection of a producer strain - a gene that causes antibiotic resistance, or a gene that completes auxotrophy. A suitable origin of replication for, for example, a plasmid vector is, for example, pM1 (der.), ColE1 (der.) And F1, pUC and F1, SV40, but is not limited to them. An element for integrating the construct into the genome of the producer may also be contained. On the

- 7 037848 example, 3 'AOX1 or 18S rRNA for yeast.

Elements for the selection of a transformant are usually understood to be a gene that causes antibiotic resistance or a gene that completes auxotrophy. When a bacterium is used as a producer, the selectable marker can be represented, for example, by a gene for resistance to ampicillin, or kanamycin, or tetracycline; yeast - for example, the LEU2, or TRP1, or URA3 genome; fungi, for example the genome of resistance to bialaphos, or hygromycin, or aureobasidin, or bleomycin; plants - for example, the genome of resistance to kanamycin or bialaphos; mammalian cells - for example, the neomycin resistance genome.

A genetic construct that provides for the synthesis of a fusion protein in the cells of a target organism, a short linear construct, must contain, in addition to the fusion gene encoding the protein according to the invention, elements for effective functioning according to its intended purpose. The development of such a genetic construct is most economically profitable to carry out using the polymerase chain reaction (PCR).

Elements for efficient functioning, for the expression of the encoded protein, are meant transcription initiation signals, promoter, translation initiation signals, startcodon and stop codon (s), transcription termination sequences, regulatory sequences. The presence of a secretory sequence is also possible.

When used as a producer of the organism Escherichia coli, the promoter can be, for example, the promoter of the lactose operon, tryptophan operon; yeast - for example, the promoter of the acid phosphatase gene, the alcohol dehydrogenase gene, the glyceraldehyde-3-phosphate dehydrogenase gene, the galactose metabolism gene; fungi, for example, the promoter of the cellobiohydrolase gene, or α-amylase, or glucoamylase, or glyceraldehyde-3-phosphate dehydrogenase, or the abp1 gene; plants - for example, the CaMV 19S RNA or CaMV 35S RNA promoter, or the nopaline synthetase gene promoter. When used as a producer of mammalian cells, for example, a promoter can be a natural promoter with its own regulatory elements (for example, CaM kinase II, CMV, nestin, L7, BDNF, NF, MBP, NSE, p-globin, GFAP, GAP43, tyrosine hydroxylase, subunit 1 of the kainate receptor and subunit B of the glutamate receptor, and others), or synthetic with regulatory sequences, to obtain the required expression pattern (the ratio of duration and expression level) of the target gene at the transcriptional level.

Translation initiation signals - Shine-Dalgarno sequence [Kapp LD, Lorsch JR The molecular mechanics of eukaryotic translation // Annual Review of Biochemistry 73/2004, 657-704] in prokaryotes and Kozak sequence in eukaryotes [Kozak M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes, Cell 44, 283-292]. When using mammalian cells, the Kozak sequence just before the ATG start codon allows for increased expression [Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J. 1997; 16 (9): 2482-2492].

The termination sequence is one of the elements that determine the stability of the mRNA. The termination sequence for eukaryotes sequentially contains a stop codon, a 3 'untranslated region with a signal and a polyadenylation site, a stop codon, due to which the stability of the mRNA is maintained and the proper termination of transcription and export of mRNA from the nucleus is carried out. The termination sequence is presented native, i.e. the initial for the cDNA of the protein used, or another, stronger one, which is presented, for example, for mammalian cells by the termination sequence of bovine growth hormone (BGH), but is not limited to it, and may contain an additional stop codon before the 3 'untranslated region. An example of a termination sequence for plant cells is that of the nopaline synthetase gene (NOS T).

Regulatory sequences are nucleotide sequences that can affect gene expression at the level of transcription and / or translation, as well as the mechanisms that ensure the existence and maintenance of the functioning of a genetic construct. Possible regulatory sequences in relation to the promoter are an enhancer to increase the level of expression through improving the interaction of RNA polymerase and DNA, an insulator to modulate the functions of an enhancer, silencers, or their fragments to reduce the level of transcription, for example, for tissue-specific expression, 5 'untranslated region before the promoter including intron. When using a silencer or an insulator as part of the structure, it is possible to regulate the expression of the target gene.

A genetic construct for protein synthesis in eukaryotic cells in one of the variants of the present invention contains from one of the above regulatory sequences, depending on the variant of the genetic construct, based on the choice of the promoter and the desired parameters of the target gene expression.

Other regulatory sequences: untranslated region downstream from the promoter, including the intron, to increase the stability of mRNA and increase the expression of the target gene.

The genetic construct for protein synthesis in eukaryotic cells according to the present invention, in one embodiment, further comprises such a regulatory element.

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For the secretion of fusion proteins according to the invention, a signal peptide suitable for the used producer or target organism is placed at the N-terminus of the polynucleotide encoding the target gene. Examples of such secretory sequences are described in the literature [for example, for E. coli - in RF patent No. 2198179, priority date 09/15/1999, for yeast - in RF patent No. 2143495, priority date 07/08/1994, US patent No. 4546082, priority date 06/17 .1982, European applications for invention Nos. 116201, 123294, 123544, 163529, 123289, application for invention of Denmark 3614/83, priority date 08.08.1983, for plants - in articles Kapila J, Rycke RD, Van Montagu M., Agenon G. (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122: 101-108, Stiefel, V., Ruiz-Avila, L., Raz, R., Valles, MP, Ghez, J., Pages, M., Martinez-lzquierdo, JA, Ludevid, MD, Langdale , JA, Nelson, T., and Puigdomenech, P. (1990). Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation. Plant Cell 2.775-793]. Moreover, it may contain a native or heterologous secretory sequence, codon optimized for the cell in which the synthesis of the fusion protein according to the invention is carried out. In one embodiment of the invention, a genetic construct for fusion protein synthesis in mammalian cells comprises the secretory sequence TPA (tissue-type plasminogen activator isoform 1 preproprotein [Homo sapiens], NCBI Reference Sequence: NP000921.1). The advantage of using the TPA secretory sequence in extensive previous clinical experience, and also in the fact that it has been shown to be highly efficient in the expression of secreted protein from various target genes.

Other elements required for the expression system to function may also be contained. Based on the state of the art, on known and obvious variants of such elements and their use, the genetic construct according to the present invention may contain any combinations that meet the above conditions, in which a hybrid gene is synthesized from the genetic construct in the cells of the producer or the target organism.

The sequence of arrangement of the described elements in the genetic construct is clear to the average person skilled in the art.

A genetic construct for expression in producer cells can be presented, depending on compatibility with the producer, with a viral, plasmid, phosmid, cosmid or other possible vector. For example, when choosing E. coli cells as a producer, the gene can be in the composition of a bacteriophage, for example, based on λ phage, or a plasmid, for example, based on pBR, or pUC, and the like. When used as a producer of a Bacillus subtilis organism, the gene can be, for example, in a pUB-based plasmid. When used as a yeast producer, the genetic construct can be represented, for example, by a plasmid based on YEp, or YRp, or YCp, or YIp. When used as a producer of mammalian cells, the genetic construct can be represented, for example, by a plasmid, for example, pVAXI, pcDNA3.1, or an adeno-associated virus, but is not limited to them.

Suitable genetic constructs for expression in cells of a targeted mammalian organism are known to those of ordinary skill in the art and described in the literature [Hartikka J., Sawdey M., Comefert-Jensen F., Margalith M., Barnhart K., Nolasco M., Vahlsing HL, Meek J., Marquet M., Hobart P., Norman J., Manthorpe M. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther. 1996 Jun 20; 7 (10): 1205-17 et al.], As well as those that can be generated by those of ordinary skill in the art using guidelines for vector elements [Cloning Vectors, ed. Pouwls et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018, Williams JA, Carnes AE, Hodgson CP. Plasmid DNA vaccine vector design: impact on efficacy, safety and upstream production. Biotechnol Adv. 2009 Jul-Aug; 27 (4): 353-70. doi: 10.1016 / j.biotechadv. 2009.02.003. Epub 2009 Feb 20. Review et al.]. Preferred genetic constructs for use in humans are vectors tested in humans, containing the elements described above with appropriate regulatory sequences, possibly modified to meet the stated criteria, thus reducing the number of studies required for drug registration. However, it is also possible to use other genetic constructs containing the required described elements.

Producers of the described hybrid proteins based on a prokaryotic or eukaryotic organism have been proposed. A procarytic producer is represented, for example, by Escherichia coli, Bacillus subtilis, eukaryotic producer - fungi, plant cells, mammals.

Producers of the described genetic constructs based on bacterial cells are proposed. A procarytic producer is, for example, Escherichia coli, Bacillus subtilis.

The examples above are given for clarity of the implementation of inventions (genetic construct, producer), but the implementation is not limited to them. At the moment, in the scientific literature (for example, textbooks on molecular biology, biotechnology, articles, for example, available at http://www.labome.ru/method/Recombinant-Protein-Expression-Vector-Host-Systems.html, specialized sites on the Internet with databases, for example, vectors) describes a variety of expression systems, as well as methods for their creation and work with them, in connection with which the specialist understands that the presented description of a group of inventions allows them to be implemented.

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Variants of the preparation for cartilage regeneration are proposed. Each variant for parenteral administration contains at least one of the described fusion proteins, as an active agent, or at least one of the described genetic constructs of one species for the synthesis of a fusion protein in the cells of the target organism, in an effective amount, as well as a physiologically acceptable carrier or buffer solution ... In the preparation, each fusion protein - either from only two components, or, when using the domain (s) for oral administration, from only three and / or four, the genetic construct - encodes one fusion protein (from 2 to 4 components). Each variant for oral administration contains from 1 fusion protein of 3 or 4 components, in an effective amount, as well as a physiologically acceptable carrier or buffer solution. Pharmaceutically acceptable carriers or buffers are known in the art and include those described in various texts, such as, for example, Remington's Pharmaceutical Sciences. Both a person and an animal, including domestic or farm animals, can act as a consumer of the drug. The oral form of the drug is administered in a special capsule that dissolves in the intestine and partly in the stomach - in an enteric coating. For example, liposomes can be used, as in the case of the recombinant oral preparation Reaferon-Lipint. Capsule fillers may also be present to further gently reduce acidity (carbonates) and protect protein. Interaction with receptors occurs quickly, and the protein does not have time to break down.

The authors of the present invention conducted laboratory studies confirming the possibility of implementing the described inventions and their effectiveness. The obtained research results are illustrated by the following examples.

List of figures, drawings and other materials

In the drawing, the components of the fusion proteins according to the invention: A - ligand to the MATN1 protein, B - EGF, C TGFe1, D - FGF2, E - flexible bridge, F - Fc-fragment of an antibody, G - polypeptide that binds to FcRn, H transferrin, I - transferrin fragment, J - IGF-1. CEA is an example of combining components in a fusion protein.

Example 1. Modeling of fusion proteins

To model proteins, the following actions were performed:

1. Search for fusion protein components

2. Building a model of the whole protein to determine the orientation of the domains

3. Building models for each domain (using sample 3D structures and ab initio)

4. Docking models using the whole protein model.

To obtain the most realistic results in an automatic mode, we used the I-Tasser algorithm for protein modeling.

Modeled fusion proteins, represented by a combination of components (Fig. 1) A, B / C / D / J, as well as those additionally containing component (s) F / G and / or H / I, components in all proteins are connected by a flexible bridge E were analyzed using the ProtParam program (http://au.expasy.org/tools/protparam.html), according to the amino acid sequences of these proteins. The following data were obtained.

The modeled hybrid proteins, represented by a combination of components (Fig. 1) A, B, connected by E, consist of 77 a.a., A, C, joined by E, - 136 a.a., A, D, joined by E, - 147 a.a., A, J, connected by E, consist of 177 a.o., and have a molecular weight of 8.4 kDa, pI 5.1.15 kDa, pI 8.74.16.3 kDa, pI 9.81, 19.2 kDa, pI 9.46, respectively.

The modeled fusion proteins, represented by a combination of components (Fig. 1) A, B, F, connected by E, consist of 316 amino acid residues, A, C, F, connected E, - 375 amino acid residues, A, D, F , connected by E, - 386 amino acids, A, J, F, connected by E, consist of 416 amino acids, and have a molecular weight of 34.7 kDa, pI 6.34, 41.3 kDa, pI 8.4, 42.7 kDa, pI 9.1, 45.5 kDa, pI 8.94, respectively. When component H is added using E, the proteins increase by 709 amino acid residues, and their molecular weight by 77.7 kDa. When added instead of component H using E component I using E, proteins increase by 27 amino acid residues, molecular weight by 2.4 kDa.

The modeled hybrid proteins, represented by a combination of components (Fig. 1) A, B, G, connected by E, consist of 126 a.a., A, C, G, joined by E, - 185 a.o., A, D, G , connected by E, - 196 amino acid residues, A, J, G, connected by E, consist of 226 amino acid residues, and have a molecular weight of 13.3 kDa, pI 5.3, 19.9 kDa, pI 8.7, 21.2 kDa, pI 9.65, 24.1 kDa, pI 9.35, respectively. When component H is added using E, the proteins increase by 709 amino acid residues, and their molecular weight by 77.7 kDa. When added instead of component H using E component I using E, proteins increase by 27 amino acid residues, molecular weight by 2.4 kDa.

The modeled hybrid proteins, represented by a combination of components (Fig. 1) A, B, H, connected by E, consist of 786 amino acid residues, A, C, H, connected E, - 845 amino acid residues, A, D, H , connected by E, - 856 amino acids, A, J, H, connected by E, consist of 886 amino acids, and have a molecular weight of 86.1 kDa, pI 6.52, 92.6 kDa, pI 7.70, 94 kDa, pI 8.3, 96.9 kDa, pI 8.25, respectively.

The modeled fusion proteins, represented by a combination of components (Fig. 1) A, B, I, co-10 037848 united E, consist of 104 a.a., A, C, I, joined by E, - 163 a.o., A , D, I, connected by E, - 174 a.o., A,

J, I, connected by E, consist of 204 amino acid residues, and have a molecular weight of 10.8 kDa, pI 5.83.17.4 kDa, pI

8.88.18.7 kDa, pI 9.89, 21.6 kDa, pI 9.55, respectively.

Based on the calculations of the above program, in all types of cells the optimal half-life of any of the proteins presented in this example is maintained in the presence of methionine at the N-terminus of such a protein. When a hybrid polynucleotide (variants) is expressed, a protein with a methionine at the N-terminus is synthesized in any cell, since translation always begins with a start codon. Further, methionine can be cleaved either naturally, for example, if the protein is secreted, as part of a secretory peptide, or it can be cleaved during protein purification, as, for example, in the case of some proteins obtained in Escherichia coli and other bacteria. Thus, a variant of the fusion protein according to the invention with or without methionine at the N-terminus is possible.

Example 2. Obtaining highly purified fusion proteins according to the invention using a prokaryotic organism

We converted the amino acid sequences of the calculated hybrid proteins into nucleotide ones, simultaneously carrying out codon optimization for expression in E. coli cells using the program http://molbiol.ru/scripts/01_19.html and adding restriction sites flanking the gene. The calculated genes were synthesized chemically.

The resulting genes were cloned into the bacterial expression vector pET28a (+) at the restriction sites flanking the target genes according to the instructions for the vector.

To create a producer strain, we used E. coli cells of the BL21 Star (DE3) strain (Invitrogen, USA), with the F-ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) genotype, containing a lysogen in the λDe3 genome and the rne131 mutation. The mutated me (rne131) gene encodes a truncated form of RNase E, which reduces intracellular destruction of mRNA, leading to an increase in its enzymatic stability. lon- and ompT-mutations in protease genes make it possible to obtain non-proteolyzed recombinant proteins in large quantities.

Prepared cells of E. coli strain BL21 with genotype F-ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) as follows. The cells were incubated at + 37 ° C overnight in 5 ml of L-broth containing 1% tryptone, 1% yeast extract, and 1% sodium chloride. The culture was diluted with fresh L broth 50-100 times and grown on a shaker at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 min. Transferred 100 ml of culture into a sterile centrifuge tube and pelleted cells at + 4 ° C at 5000g for 10 min. The supernatant was discarded, the cells were resuspended in deionized water in the original volume, followed by centrifugation. The washing procedure was repeated three times. After washing, the cell pellet was resuspended in a small volume of deionized water and centrifuged for 30 s at 5000 rpm in a microcentrifuge.

The transformation of competent cells was carried out by electroporation. For this, 1 μL of a tenfold diluted ligase mixture was added to 12 μL of competent cells, mixed, and electroporation was performed on an Eporator electroporator (Eppendorf, Germany) in sterile electroporation cuvettes (Eppendorf, Germany), with a volume of 100 μL, a 1 mm slit, with an electric pulse with a voltage of 1.7 kV with a duration of 5 ms.

After transformation, cells were incubated in SOC medium (2% bacto-tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) for 40 min. at + 37 ° C. 10-100 μl of cell suspension was plated on selective LB medium (Gibko BRL, USA) containing kanamycin (100 μg / ml) to select clones containing plasmids (producer strains).

The grown E. coli colonies were checked for the presence of plasmids with the insertion of the target gene. A clone of cells containing the desired plasmid DNA was considered a fusion protein producing strain. Thus, the producing strains of the fusion proteins according to the invention were obtained.

For the cultivation of the obtained producer strains, a standard agar LB medium containing kanamycin at a concentration of 100 μg / ml and glucose at a concentration of 1% was used to block nonspecific expression.

Expression was induced when the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm. 0.2% lactose was used as an inducer (Studier, 2005).

For autoinduction of expression by the method of Studier (Studier, 2005), we used PYP-5052 medium consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na ₂ HPO ₄ , 50 mM K2HPO4, 25 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.5% glycerol, 0.05% glucose, and 0.2% lactose.

A single colony of the producer strain was inoculated into PYP-5052 medium containing kanamycin at a concentration of 100 μg / ml. Fermentation was carried out at + 37 ° C in a thermostated rotary-type shaker at 250 vol. min. within 20 h until there was no significant change in OD ₆₀₀ for 1 h. An aliquot of cells was taken for analysis of the expression of the gene encoding the fusion protein by electrophoresis in PAGE, and the remaining biomass was precipitated by centrifugation at 9000g.

The precipitated cells were lysed using 3 sonication cycles of 30 s each with a break of 2 min for

- 11 037848 ice. Then, the destruction of inclusion bodies was carried out by incubation with lysis buffer containing 500 mM sodium phosphate buffer, pH 8.0, 6M guanidine hydrochloride, 500 mM sodium chloride, for an hour. To the cells harvested by centrifugation from 50 ml of culture was added 8 ml of lysis buffer.

The column containing Ni-NTU Sepharose was pre-equilibrated with loading buffer (500 mM sodium phosphate buffer, pH 8.0, 8 M urea, 500 mM sodium chloride, 10 mM imidazole). The destroyed inclusion bodies were applied to the column. The column was then washed with two volumes of loading buffer (500 mM sodium phosphate buffer, pH 8.0, 8 M urea, 500 mM sodium chloride, 10 mM imidazole). The column was then washed with three volumes of wash buffer (500 mM sodium phosphate buffer, pH 8.0, 8 M urea, 500 mM sodium chloride, 30 mM imidazole). The protein was eluted with 5 ml of elution buffer (500 mM sodium phosphate buffer, pH 8.0, 8M urea, 500 mM sodium chloride, 200 mM imidazole). Fractions of 1 ml were collected, analyzed by electrophoresis in 12% PAGE-DDs-Na, fractions with the target protein were combined, the protein concentration in them was determined by the Bradford method.

Received protein preparations with a purity of about 97-98%, according to SDS-PAGE, the concentration of the fusion protein in each preparation was 1-2 mg / ml.

Example 3. Obtaining highly purified hybrid proteins according to the invention using a eukaryotic organism

3.1. Obtaining highly purified hybrid proteins using yeast cells.

We converted the amino acid sequences of the calculated hybrid proteins into nucleotide ones, simultaneously carrying out codon optimization for expression in Pichia pastoris yeast cells using the program http://molbiol.ru/scripts/01_19.html and adding the gene flanking regions to obtain the secreted protein, according to the instructions for the vector for cloning. The calculated genes were synthesized chemically.

The resulting genes were cloned into the eukaryotic expression vector pHIL-S1 according to the instructions for the vector.

Yeast cells were prepared for transformation. Cultivation and freezing of cells of Pichia pastoris strain SMD1163, defective in several yeast proteases, was carried out, which ensures the stability of the secreted protein. The cells were seeded under sterile conditions on agar in YPD medium (1% yeast extract, 2% peptone, 2% glucose, 1 mM dithiothreitol), cultured at 30 ° C, then subcultured into suspension and cultured for 16 h. resuspended in YPD medium supplemented with 15% glycerol and frozen at -86 ° C. To obtain competent cells, cell colonies were preliminarily grown on an agar plate in YPD medium at 30 ° C for two days. Then the contents of one colony were grown in 10 ml of YPD medium at 30 ° C for 16 h. The suspension was diluted in YPD to OD ₆₀₀ 0.2 and a final volume of 10 ml, and the culture was grown to OD ₆₀₀ 0-8 for 4 h. The cell suspension was centrifuged 5 min at 500 g, the supernatant was poured out, resuspended in 10 ml of solution I from the EasyComp Transformation Kit, centrifuged again, and the pellet was resuspended in solution I. temperature of -90 ° C before use.

The EasyComp Transformation Kit included in the Pichia Easy Select Kit (Invitrogen) was used for transformation; the reaction was carried out according to the instructions for the kit. The resulting cell suspension was plated into a sterile dish on an agar gel prepared on YPD medium supplemented with 1M sorbitol and the antibiotic ampicillin at a final concentration of 100 μg / ml. After 3 days, several dozen colonies were obtained per dish. Cells from the grown colonies were transferred onto a plate with MMD (minimal medium dextrose) agar and cultured for 2 days at 30 ° C.

The cells of the colonies grown on the selective medium were transferred into flasks and cultured in 5 ml of MGY medium on a shaker (250 rpm) for 1 day until OD ₆₀₀ 5. After that, the cells were pelleted by centrifugation at 3000 g for 10 min. Control of the target gene expression was performed by SDS-PAGE.

After the cells were precipitated, the culture medium was filtered (pore diameter 45 μm), then Tris-HCl pH 6.0 was added to a final concentration of 20 mM. The culture medium containing the fusion protein was concentrated 5-10 times using Millipore concentrators for proteins with a molecular weight of more than 10 kDa.

After concentration, the fusion protein preparation was heated in a water bath to boiling (t = 100 ° C) and boiled for 2 min, after which it was centrifuged at 4 ° C, 15000xg for 15 min.

Ion exchange chromatography was carried out on CM-Sepharose. The column with CM-Sepharose was equilibrated with a buffer containing 20 mM Tris-HCl pH 6.0. The fusion protein preparation was applied at a rate of 60 ml / h. The column was washed with 20 mM Tris-HCl pH 6.0; 20 mM Tris-HCl pH 6.0, 200 mM NaCl. Elution was performed with 20 mM Tris-HCl pH 6.0, 1 M NaCl, and fractions of 1 ml were collected.

The preparation of the obtained fusion protein was diluted 2 times, phosphate pH 8.0 was added to a concentration of 50 mM, and applied to the column. After washing the column with loading buffer, ballast proteins were removed by washing with 20 mM imidazole solution in the same buffer. The protein was eluted with a solution containing 200 mM imidazole.

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As a result, preparations of hybrid proteins with a purity of over 95% were obtained. The presence of bands on the electropherograms corresponding to the molecular weight of the target proteins was revealed. The resulting strains are characterized by a high level of expression of targeted proteins.

3.2. Obtaining highly purified fusion proteins using mammalian cells.

The TPA signal sequence (tissue-type plasminogen activator isoform 1 preproprotein [Homo sapiens], NCBI Reference Sequence: NP_000921.1) was placed at the N-terminus of the amino acid sequence of each fusion protein. We converted the amino acid sequences of the calculated fusion proteins into nucleotide ones, simultaneously performing codon optimization for expression in mammalian cells (CHO) in manual mode and adding restriction sites flanking the gene and the Kozak sequence. The calculated genes were synthesized chemically.

The synthesized gene was cloned into the pcDNA3.1 (+) vector according to the instructions for the vector. On the basis of E. coli DH10 B / R cells, a producing strain of this plasmid DNA was created, as described in clause 4.1.1. protocol.

Transfection of mammalian cells with the created plasmids was carried out by the method of calcium phosphate precipitation.

To transform mammalian cells (CHO) with plasmid DNA, the cells were seeded in 12-well plates (Costar, USA) with a seeding density of 5x10 ⁴ cells / cm ² . The next day, the culture medium was changed to synchronize cell division. After 3 h, calcium phosphate-precipitated plasmid DNA was added to the cells. To prepare a precipitate, 250 μl of a solution containing 50 μg of DNA in 250 mM CaCl ₂ was slowly mixed with 250 μl of a solution (1.64% NaCl, 1.13% HEPES pH 7.12, and 0.04% Na ₂ HPO ₄ ). After 24 h of incubation at 37 ° C in an atmosphere of 5% CO _{2, the} medium was replaced with a similar medium containing 100 μg / ml of neomycin for selection of clones containing plasmids with the insertion of the target gene and, therefore, expressing hybrid proteins, the selection was carried out for 20 days, in the wells containing live cells, the medium was changed (in this case, the previous culture medium was not poured out, but was used to determine the amount of secreted proteins by ELISA), and after another day the cells were removed from the substrate and analyzed for the expression of the transformed genes. Transfection efficiency was analyzed using an EPICS XL Beckman Coulter flow cytometer (Beckman Coulter, USA).

The level of fusion proteins in the culture medium of the obtained stable transfect line CHO was assessed using standard solid phase ELISA.

As a result of cloning, stable CHO transfectomes were obtained, which were accumulated for cryopreservation and production of an experimental batch of fusion proteins. The productivity of transfected CHO expressing fusion proteins was 420-540 μg / 10 ⁷ cells / day.

Cultivation of producer cells was carried out using a BIOSTAT® Bplus bioreactor and an autoclaved IMDM medium supplemented with 45 g of DFBS (0.5%) and 25.8 g (100 mM) of zinc heptahydrate (ZnSO ₄ x 7H2O) per 9 liters of medium. The operating mode was set: temperature 37 ° C, pH 6.9-7.2, oxygen concentration 50% of air saturation. After reaching the specified mode, the bioreactor was inoculated, for which the inoculum was introduced into it under aseptic conditions. The cultivation time was 3 days.

At the end of the cultivation, the culture fluid was filtered through a sterile Sartopure capsule (Sartorius, Germany), with a pore diameter of 1.2 μm, at a rate of 1 L / min. Then the clarified liquid was concentrated on a Viva Flow 200 system (Sartorius, Germany) using a filter. Concentration was carried out until a total volume of 200 ml was reached.

Chromatographic purification was carried out in two stages using sterile solutions. At the first stage, a BioLogic DuoFlow Pathfinder (Bio-Rad) system with an automatic BioFract fraction collector and a YMC TriArt semi-preparative chromatographic column, 250x4.6 mm, C18 sorbent were used. Before starting work, the column was equilibrated using 200 ml of buffer (1 kg of water for injection and 1 g of trifluoroacetic acid) in manual mode through a chromatograph pump at a rate of 2 ml / min.

The prepared material in a volume of 200 ml was introduced into the chromatograph through the chromatograph pump at a rate of 0.5 ml / min. Elution was performed with a buffer (2 kg of acetonitrile, 2 g of trifluoroacetic acid) at a rate of 0.5 ml per minute. Collected the fraction at the maximum absorption at 260 nm. The fraction volume was approximately 500 ml.

The second stage of purification was carried out using a BioSil SEC 125-5 gel chromatography column, 300x7.8 mm. The column was pre-equilibrated with 0.02 M PBS buffer. The resulting material was introduced into the chromatograph through the chromatograph pump at a rate of 0.5 ml / min. Elution was performed with a buffer (0.6 M NaCl solution) with a concentration gradient from 0.1 to 0.6 M. A fraction was collected with an absorption at A280 nm of at least 3.4 optical units. The fraction was collected in vials. The volume of the resulting solution for each protein preparation was approximately 1 L with a fusion protein concentration of 2-2.7 mg per 1 ml.

The fusion proteins according to the invention can also be obtained using other cells

- 13,037848 mammals, eg HEK293, COS.

3.3. Obtaining highly purified hybrid proteins using plants.

We converted the amino acid sequences of the calculated hybrid proteins into nucleotide ones, simultaneously carrying out codon optimization for expression in Nicotiana benthamiana cells using the program http://molbiol.ru/scripts/01_19.html and adding the gene flanking regions according to the instructions for the cloning vector. The calculated genes were synthesized chemically and cloned into the pTRV1 eukaryotic expression vector. It is also possible to use a viral vector (for example, described in the article Komarov T.V., Skulachev M.V., Zverev A.S., Shvarts AM, Dorokhov Yu.L., Atabekov I.G. (2006) New virus vector for efficient production of target proteins in plants Biochemistry, 71 (8), 1043-1049).

The resulting vector was introduced into the Agrobaterium tumefaciens GV3101 strain, which was used for infiltration of N. benthamiana leaves. The resulting Agrobaterium tumefaciens strain carrying the hybrid gene was cultured for 12 h at 30 ° C in a shaker. Cells (1.5 ml) was pelleted by centrifugation (4000g, 5 min), the pellet was resuspended in buffer (1.5 ml of 10 mM MgCl _2, 10 mM MES (pH 5.5)), OD ₆₀₀ was adjusted to 0.2. A suspension of Agrobacteria was applied with a syringe without a needle to the leaves of growing N. benthamiana plants. The maximum level of protein synthesis was observed on days 7-11 after infiltration.

The expression of fusion proteins in the leaf cells of producing plants was analyzed using SDS Na electrophoresis in PAGE. The leaf fragment was triturated in buffer (10 mM KCl, 50 mM Tris pH 8.0, 5 mM MgCl ₂ , 10 mM β-mercaptotanol, 0.4 M sucrose, 10% glycerol) on day 10 after infection. The obtained extract was subjected to centrifugation (14000g, 10 min), the pellet and supernatant were analyzed using SDS-PAGE. The electrophoretogram revealed proteins corresponding in molecular weight to the hybrid proteins according to the invention in the membrane fraction of cells. In the control, plants that did not undergo transformation, the corresponding proteins were not identified. The protein yield was about 12-14% of the fraction of insoluble proteins.

Based on the results obtained, the claimed hybrid proteins can be obtained using both prokaryotic and eukaryotic cell systems, a highly purified preparation of each protein can be obtained using various types of protein purification. The isolation and purification conditions given were selected experimentally and may vary within the values known to the average person skilled in the art.

Example 4. Obtaining highly purified genetic constructs according to the invention

We converted the amino acid sequences of the calculated hybrid proteins into nucleotide ones, simultaneously carrying out codon optimization for expression in mammalian cells using the program http://molbiol.ru/scripts/01_19.html and adding the restriction sites flanking the gene and the Kozak sequence. The calculated genes were synthesized chemically.

4.1. Obtaining plasmid DNA encoding a fusion protein according to the invention (variants)

4.1.1. Creation of a plasmid DNA producer strain

The resulting genes were placed into eukaryotic expression vectors pVAX1 (Invitrogen) or pcDNA3.1 + (Invitrogen) at restriction sites flanking the target genes according to the instructions for the vector.

Coli cells of DH10B / R strain (F-mcrA, Δ (mrrhsdRMS-mcrBC), φ80dlacZΔM 15, ΔlacX74, deoR, recA1, endA1, araD139, Δ (ara, leu) 769, galU, galK -, rpsL, nupG) obtained plasmid DNA by electroporation using a MicroPulser electroporator (BioRad). This strain does not contain methylase, which makes it possible to minimize the possibility of mutations in DNA, including in the gene cloned in a plasmid maintained in this strain. To 12 μl of competent cells, 1 μl of dialyzed ligase mixture was added, placed between the electrodes of the pore cell, and treated with a current pulse.

After transformation, the cells were placed in 1 ml of SOC medium (2% bactotrypton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) and incubated for 40 min at + 37 ° C.

Identification of clones of E. coli cells containing the obtained plasmid DNA was carried out on a selective medium containing LB-agar, 50 μg / ml of kanamycin or ampicillin, respectively.

Plasmid DNA was isolated from the grown clones. Isolation of plasmid DNA was performed using the Wizard Minipreps DNA Purification System kit (Promega, United States). Purified recombinant plasmid DNA was verified by sequencing.

The cloned fragments were sequenced according to the Sanger method using the Applied Biosystems BigDye® Terminator (BDT) v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the instructions supplied with it. For labeling the reaction products, ddNTPs labeled with a fluorescent dye were used, with each ddNTP corresponding to its own dye. Unlabeled plasmid-specific primers were used for sequencing. A PCR reaction was carried out, then the reaction mixture was purified from free labeled ddNTPs according to the instructions for the BigDye X-14 kit 037848

Terminator Purification Kit (Applied Biosystems, USA) and the sequencing reaction products were separated using an Applied Biosystems 3500 / 3500xL Genetic Analyzer (Applied

Biosystems, USA) and 3500 / 3500xL Genetic Analyzer Polymer POP-6 ™ reagent (Applied Biosystems,

USA).

The results of the separation of the sequencing reaction products were recorded by laser scanning and detection of four fluorescent dyes included in all types of ddNTPs.

Computer analysis of DNA sequences was performed using a personal computer using the Chromas and BioEdit programs. The nucleotide sequences of the studied DNA fragments were aligned relative to the calculated ones, the identity of the synthesized fragments with the calculated ones was demonstrated. As a result, clones of E. coli cells were selected containing full-length sequences of target genes as part of plasmids, DNA sequences encoding fusion proteins.

4.1.2. Production of plasmid DNA encoding a fusion protein (variants)

A separate colony of E. coli grown on LB-agar in a Petri dish supplemented with kanamycin or ampicillin was placed in 10 ml of selective medium. The cells were grown for 12 h at + 37 ° C under constant stirring (250 rpm). The resulting cells were harvested by centrifugation at 4000g. Further isolation and purification of plasmid DNA was carried out using the EndoFree Plasmid Mega Kit (Qiagen), which allows to obtain pyrogen-free DNA. The isolated plasmid DNA was analyzed by electrophoresis in 0.8% agarose gel, its concentration was measured using fluorimetry. The yield of plasmid DNA ranged from 3.1 mg to 4.7 mg from 1 liter of culture medium.

4.2. Obtaining a vector based on an adeno-associated virus encoding a fusion protein according to the invention (variants)

The resulting genes were placed into a vector based on the adeno-associated virus pAAVK-EF1a-MCS (System Biosciences (SBI)), on the basis of which a producing strain of this vector was created using E. coli cells (RecA ^- ), then the vector was isolated for use in mammals, all according to the instructions for the vector. The output of the vector was from 2 to 3.2 mg from 1 liter of nutrient medium.

4.3. Obtaining a short linear construct encoding a fusion protein according to the invention (variants)

To develop a short linear design, the one obtained in clause 4.1 was used. plasmid DNA. Using specific primers and PCR, a fragment of plasmid DNA was amplified containing transcription initiation signals, a promoter, translation initiation signals, a start codon, a fusion gene, 1 or 2 stop codons, transcription termination sequences, and regulatory sequences.

Amplification of this sequence was carried out in a volume of 50 μl, in thin-walled polypropylene tubes with a volume of 650 μl containing 5 μl of 10-fold Taq buffer (700 mM TrisHCl, pH 8.6 / 25 ° С, 166 mM (NH ₄ ) ₂ SO ₄ ), 5 μl MgCl ₂ (1.25 mM), 1 μl dNTP, 31.5 μl water, 1 μl forward and 1 μl reverse primers, 5 μl plasmid DNA, and 0.5 μl Taq polymerase (Fermentas, Lithuania).

The reaction mixture was heated for 5 min at 95 ° C to denature the DNA. To prevent evaporation, 30 μL of Bayol F mineral oil (Sigma, USA) was layered on the reaction mixture with a volume of 50 μL. The amplification reaction was carried out in a C1000 Thermal Cycler (Bio-Rad, USA). 35 cycles were carried out: 95 ° С - 20 s, 50-62 ° С (depending on the dimensions) - 20 s, 72 ° С - 1 min. To complete the resulting DNA strands, an additional cycle was performed: 5 min at 72 ° C.

The PCR result was analyzed by agarose gel electrophoresis. If the result was positive, preparative electrophoresis was performed.

The amplified DNA fragments were concentrated and purified by preparative electrophoresis in 1.2% agarose gel (Gibko BRL, USA). After PCR, a sample of the mixture was mixed with a six-fold buffer (0.25% bromophenol blue, 30% glycerol) (ThermoScientific, USA) and applied to the gel, 18 μL per well. Electrophoresis was performed in a horizontal apparatus in TAE buffer (40 mM Tris-acetate, 2 mM EDTA pH 8.0, 0.5 μg / ml ethidium bromide) at a voltage of 5-10 V / cm. The result of DNA separation was recorded in transmitted UV light (302 nm) of a Macrovue transilluminator (LKB, Sweden). The length of the amplified fragment was determined from the logarithmic dependence of the DNA mobility on the length of the fragments in the marker. The proprietary mixture of DNA fragments GeneRuler 1000 bp DNA Ladder (Fermentas, Lithuania) was used as markers. A section of agarose containing a DNA band of the required size was excised, and the DNA fragment was purified using the DNA & Gel Band Purification Kit (GE Healthcare, UK) according to the instructions. An isolated mammalian short linear construct was used.

Example 5. Determination of the regenerative effect of fusion proteins according to the invention

Collagen-induced arthritis in rats is a model of human rheumatoid arthritis. This type of arthritis in rats was induced by the introduction of native heterologous type II chicken collagen as part of Freund's incomplete adjuvant. Type II collagen is the target of this autoimmune attack. In collagen-induced arthritis, the leading role in the implementation of effector reactions belongs to autoantibodies to collagen (detailed description of the models: Kleinau S., 1991, Gromyko, Gritsuk,

2012).

Investigated hybrid proteins according to the invention, with the introduction of a single protein and with the introduction of a mixture of proteins, with oral administration, as well as with parenteral administration. The results were evaluated by analyzing histological sections of the joints, microscopy.

5.1. Investigation of the regenerative potential of a drug based on one hybrid protein (variants) for parenteral administration

5.1.1. Introduction to periarticular tissue

After the formation of arthritis, rats were injected with a fusion protein (variants), based on components A, B / C / D / J, and in some variants additionally F / G and / or H / I, the components in the protein variants are presented in different order and connected by the component E, all components are shown in FIG. 1, or a mixture based on them, in an amount of 20 μg of protein locally - in the periarticular tissues, mainly in the muscles, which corresponds to a human dose of 0.1 mg. The introduction was carried out daily for seven days. Starting at day 6, the morphology of the joints was analyzed for 21 days. Saline was used as a negative control. A reliable picture of cartilage recovery was obtained using all the studied fusion proteins, compared with the control, while the best performance among the groups with the introduction of one fusion protein was observed in the AEJ group, the groups with the introduction of two fusion proteins - in the AEJ + JEA group, with a small margin - in the group AEC + AEB + DEA + AEJ, the components are shown in FIG. 1. Hybrid proteins, additionally containing component (s) from F / G and / or H / I, generally had an effect comparable to that of proteins of a similar structure that do not contain these domains, that is, caused the regeneration of damaged cartilage, less pronounced in the case of containing two additional domains.

5.1.2. Systemic introduction

After the formation of arthritis, rats were injected with a fusion protein (variants) based on components A, B / C / D / J, and in some variants additionally F / G and / or H / I, the components in the variants of the protein are presented in different order and connected by component E , all components are shown in FIG. 1, or a mixture based on them, in an amount of 20-30 μg of protein systemically - into the tail vein, which corresponds to a dose for humans of 0.2-0.3 mg. The introduction was carried out daily for seven days. Starting at day 6, the morphology of the joints was analyzed for 21 days. Saline was used as a negative control. A reliable picture of cartilage recovery was obtained using all the studied fusion proteins, compared with the control, while the best performance among the groups with the introduction of one fusion protein was observed in the JEA group, the groups with the introduction of two fusion proteins - in the CEA group with methionine at the N-terminus protein (without formyl) + JEA, with a small margin - in the CEA + BEA + JEA group, the components are shown in Fig. 1. When using fusion proteins containing additional component (s) from F / G and / or H / I, a slightly less pronounced regeneration of cartilage was observed as a result of the experiment, however, after the end of the experiment, a longer circulation of these proteins in the body was observed, which may have caused continuation of their action on the restoration processes in the cartilage.

Less pronounced cartilage regeneration was observed as a result of this experiment, compared to that in the experiment described in paragraph 5.1.1.

5.2. Investigation of the regenerative potential of a drug based on one genetic construct (variants) for parenteral administration

5.2.1. Introduction to periarticular tissue

After the formation of arthritis, rats were injected with from 1 of the created genetic constructs based on plasmid DNA, or an adeno-associated virus, or a linear fragment from which a hybrid protein based on components A, B / C / D / J was synthesized in animal cells, and in some variants additionally F / G and / or H / I, the components in the variants of the protein are presented in different order and connected by the component E, all components are shown in FIG. 1, in an amount of 50 μg of DNA locally - in periarticular tissues, mainly in muscles, which corresponds to a human dose of 1 mg. The introduction was carried out once every seven days, a total of five times. After the third injection, the morphology of the joints was analyzed for 21 days. The same genetic construct, which does not contain the hybrid gene, was used as a negative control. A reliable picture of cartilage restoration was obtained using all the studied genetic constructs (DNA), compared with the control, while the best indicators among the groups with the introduction of one genetic construct were observed in the groups based on pVAX plasmid DNA encoding the hybrid AEJ protein, as well as on the basis of linear constructs encoding the fusion protein AEDEF, with a small margin - in the group based on the adeno-associated virus encoding the FEBEA protein, groups with the introduction from two genetic constructs - in the group based on the plasmid DNA pcDNA3.1 encoding fusion proteins AEJ + JEA (gene with signal secretion at the N-terminus), with a small margin - in the group based on plasmid DNA pVAX encoding fusion proteins AEC + AEB + DEA + JEA, the components are shown in Fig. 1. Genetic constructs encoding fusion proteins, additionally containing the above-mentioned fourth component, generally had an effect comparable to that of proteins of similar structure that did not contain these domains, that is, they caused regeneration of damaged cartilage, but less pronounced. It was also revealed that the synthesized fusion proteins were detected longer in the serum of animals after the end of the experiment with the introduction of genetic constructs encoding one transport domain from F / G / H / I. As a result of the introduction of the linearized vector, slightly lower rates of regeneration were observed.

5.2.2. Systemic introduction

After the formation of arthritis, the rats were injected with the created genetic constructs based on plasmid DNA, or an adeno-associated virus, or a linear fragment, from which a fusion protein based on components A, B / C / D / J was synthesized in animal cells, and in some variants additionally F / G and / or H / I, the components in the protein variants are presented in different order and connected by the component E, all components are shown in FIG. 1, or a mixture based on such genetic constructs, in an amount of 50 μg of the genetic construct systemically into the tail vein, which corresponds to a human dose of 1 mg. The introduction was carried out once every seven days, a total of five times. After the third injection, the morphology of the joints was analyzed for 21 days. The same genetic construct, which does not contain the hybrid gene, was used as a negative control. A reliable picture of cartilage restoration was obtained using all the studied genetic constructs compared to the control, while the best indicators among the groups with the introduction of one genetic construct were observed in the groups based on the adeno-associated virus encoding the JEA protein, the groups with the introduction of two genetic constructs - in the group on based on plasmid DNA encoding CEA + JEA proteins, with a small margin - in the group based on linear fragments encoding CEA + BEA proteins (with a signal sequence at the N-terminus) + JEA, the components are shown in Fig. 1. Slightly less pronounced cartilage regeneration was observed as a result of this experiment as compared to that in the experiment described in paragraph 5.2.1 ..

Genetic constructs encoding fusion proteins, additionally containing the F / G and / or H / I component (s), had an effect comparable to that of proteins of a similar structure that do not contain these domains, that is, they caused the regeneration of damaged cartilage, but the synthesized fusion proteins take longer were detected in the serum of animals after the end of the experiment.

5.3. Study of the regenerative potential of a preparation based on a single fusion protein (variants) when administered orally

Fusion protein (variants), represented by components A, B / C / D / J, F / G and / or H / I, in different order, connected by component E, the components are shown in Fig. 1, or a mixture based on such fusion proteins, was placed in an enteric coating and administered to rats after the formation of arthritis in an amount of 2-3 mg of protein orally, which corresponds to a human dose of 20-30 mg. The introduction was carried out daily for 7-30 days. Starting at day 10, the morphology of the joints was analyzed for 21 days. Water was used as a negative control. As a positive control, a preparation of chondroitin sulfate was used, 4 mg was injected, which corresponds to a human dose of 750 mg, 2 times a day for 3 weeks.

A reliable picture of cartilage recovery was obtained using all the studied fusion proteins, compared with the control, while the best indicators among the groups with the introduction of one fusion protein were observed in the groups GEAEB / C / D / J, GEB / C / D / JEA, groups with the introduction from two fusion proteins in the IEJEA + AEBEH group, the components are shown in FIG. 1. It was found that when used in conjunction with a three-component or four-component (including the transport domain) two-component fusion protein (not including the transport domain), the difference in the results is insignificant, compared with the introduction of a protein of three or four components.

Cartilage regeneration was observed as a result of this experiment, comparable to that observed in experiment 5.1.1. In addition, long-term circulation of fusion proteins containing the transport domain in the blood was observed. The use of one or another enteric coating did not significantly affect the results of the study.

We also conducted a similar study on rabbits that underwent surgical tearing of the menisci. The results of this study correlate with the results obtained in a model study of collagen-induced arthritis in rats.

Thus, the possibility of obtaining hybrid proteins, polynucleotides, genetic constructs, producers, preparations for cartilage regeneration (all variants) has been proved. It was also shown that the developed hybrid proteins reliably have a regenerative effect on damaged cartilage, both directly introduced and with the introduction of genetic constructs encoding them. In all studies, an increase in the number of chondrocytes in previously damaged joints was observed, which, most likely, caused cartilage regeneration. This allows us to conclude that in other types of cartilage damage, the proposed invention (variants) will allow for their regeneration.

Claims (9)

CLAIM 1. A fusion protein comprising a ligand to the MATN1 protein, characterized by the amino acid sequence SEQ ID NO: 1, and a growth factor from EGF, TGF, FGF, IGF, characterized by the amino acid sequence SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4 or SEQ ID NO: 5, respectively; the components of the construct are connected by a flexible bridge characterized by the amino acid sequence SEQ ID NO: 6. 2. A fusion protein comprising a ligand to the MATN1 protein, characterized by the amino acid sequence SEQ ID NO: 1, a growth factor from EGF, TGF, FGF, IGF, characterized by the amino acid sequence SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, respectively, as well as the Fc fragment of an antibody, characterized by the amino acid sequence SEQ ID NO: 7, or a polypeptide that binds to FcRn, characterized by the amino acid sequence SEQ ID NO: 8, and / or transferrin, characterized by the amino acid sequence SEQ ID NO: 9, or a fragment thereof characterized by the amino acid sequence SEQ ID NO: 10; the components of the construct are connected by flexible bridges characterized by the amino acid sequence SEQ ID NO: 6. 3. A polynucleotide encoding a fusion protein according to claim 1 or 2, codon optimized for expression in the cells of the producer or target organism. 4. A genetic construct for the synthesis in the cells of a producer or a target organism of a hybrid protein according to claim 1 or 2, comprising a polynucleotide according to claim 3 and elements allowing to realize the specified purpose. 5. Producer of the fusion protein according to claim 1 or 2. 6. Producer of a genetic construct according to claim 4 based on a bacterial cell. 7. A preparation for cartilage regeneration containing at least one fusion protein according to claim 1 and / or 2 as an active agent in an effective amount, as well as a physiologically acceptable carrier or buffer solution for parenteral administration. 8. A preparation for cartilage regeneration containing at least one genetic construct for synthesis in cells of a target organism according to claim 4 as an active agent, in an effective amount, as well as a physiologically acceptable carrier or buffer solution for parenteral administration. 9. A preparation for cartilage regeneration containing at least one fusion protein according to claim 2 as an active agent in an effective amount, as well as a physiologically acceptable carrier or buffer solution for oral administration, the preparation is enclosed in an enteric coating.